Molybdopterin is the essential, invariant component of the cofactors of all mononuclear molybdenum and tungsten-containing enzymes. Most prokaryotic enzymes contain dinucleotide forms of molybdopterin complexed with GMP, AMP, CMP or IMP. The most elaborate form of the cofactor is bis(molybdopterin guanine dinucleotide)-Mo, first identified in R. sphaeroides dimethyl sulfoxide reductase (DMSOR) which contains no other cofactor. R. sphaeroides biotin sulfoxide reductase (BSOR) and E. coli trimethylamine-N-oxide reductase (TMAOR) also contain bis(molybdopterin guanine dinucleotide)-Mo as their sole cofactor, and all three enzymes have been cloned and successfully expressed in E. coli. In collaboration with crystallographers at other universities, the X-ray crystal structure of DMSOR has been determined. BSOR is of particular interest since it is the only enzyme among the three that uses NADPH as an electron donor. Detailed steady state and stopped-flow kinetic analysis will be used to compare the mechanism of action of both wild type and site-directed mutagenesis-derived variants of DMSOR, BSOR TMAOR with their native electron donors. Giant strides have been made in our studies of the biosynthesis of the molybdenum cofactor in E. coli, both at the mechanistic and structural level. All of the biosynthetic proteins have been expressed and purified, and in vitro assay systems have been developed for the formation of the dithiolene moiety of the cofactor, incorporation of molybdenum, and formation of the guanine dinucleotide form of the cofactor. In collaboration with Dr. Hermann Schindelin at SUNY Stow Brook, the crystal structures of all of the cofactor biosynthesis proteins have been determined with the exception of MoaA, the first protein in the pathway. Along with the in vitro assays, these structures generate an unlimited vista for future structure-function studies of each step in the pathway using variants generated by site-directed mutagenesis. Optimization of MoaA expression and purification will be undertaken to generate sufficient quantities of soluble protein for structural determination and for studies of the formation of precursor Z, the first intermediate in the pathway. A complete understanding of cofactor biosynthesis is of great importance since molybdenum cofactor deficiency in humans causes severe neurological defects and generally results in infant fatality.